Pakistan Veterinary Journal ISSN: 0253-8318 (PRINT), 2074-7764 (ONLINE) DOI: 10.29261/pakvetj/2021.019 SHORT COMMUNICATION

Antibacterial Activity of Medicinal Flowers against Multi Drug Resistant E. coli

Iqra Nazir1, Masood Rabbani1*, Ali Ahmad Sheikh1, Sehrish Firyal2, Sohail Raza1 and Muhammad Ilyas Riaz1

1Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan 2Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan *Corresponding author: [email protected]

ARTICLE HISTORY (20-522) ABSTRACT

Received: October 12, 2020 The antibiotic resistance is creating a serious challenge to treat diseases especially Revised: December 23, 2020 against multidrug resistant (MDR) E. coli. The present study was conducted to Accepted: January 08, 2021 Published online: January 26, 2021 evaluate the antibacterial activity of seven different flowers ethanolic extracts Key words: named as Achillea millefolium, Bombax ceiba, Chrysanthemum cinerarifolium, Antibacterial activity Hyssopus officinalis, Rosa damascena Miller, Taraxacum officinale Weber and E. coli Woodfordia fruticosa against MDR E. coli through spot test and minimum Medicinal flowers inhibitory concentrations (MIC) test. The results showed all the seven extracts have Multidrug resistant (MDR) significant antibacterial activity against MDR E. coli and control. The MIC of Rosa damascena Miller, Bombax ceiba was 3.125 mg/ml and Taraxacum officinale Weber was 12.5 mg/ml for MDR E. coli. Similarly, Achillea millefolium, Hyssopus officinalis, Chrysanthemum cinerarifolium MIC value was 25 mg/ml and 12.5 mg/ml for Woodfordia fruticosa Kurtz. The current study reveals that these flowers possess strong antibacterial activity and thus can be used as potential antimicrobial agent against various resistant bacterial pathogens.

©2021 PVJ. All rights reserved To Cite This Article: Nazir I, Rabbani M, Sheikh AA, Firyal S, Raza S and Riaz MI, 2021. Antibacterial activity of medicinal flowers against multi drug resistant E. Coli. Pak Vet J, 41(1): 166-168. http://dx.doi.org/10.29261/pakvetj/2021.019

INTRODUCTION natural intestinal flora of almost all warm blooded animal, carry drug resistance genes due to irrational use of In recent years, antimicrobial resistance (AMR) has antibiotics. With increasing trend of AMR, experiments become one of the most important public health concern are being under way to use alternatives to antibiotics. The of this century. The infectious diseases are caused by natural ingredients have least side effects and are more bacteria, viruses, parasites and fungi. Among these, effective against pathogens as compared to bacteria are a major problem of some common and severe chemosynthetic agents. The plant extracts have wide infections. The irrational uses of antibiotics have spectrum of active compounds like terpenoids, flavonoid, developed resistance against the drugs that are commonly coumarin, tannin, saponin, alkaloids, glucosides, essential used to treat bacterial infections. The bacteria causing the oils and polysaccharides. The natural constituents can be common or lethal infections have developed resistance to derived from any part of the plants are a gift to cure existing and new antibiotics coming to the market that diseases and have vital role in health development (Karole leads to a global health crisis (Prestinaci et al., 2015). et al., 2017). It has been found that natural products are Antibiotics put a selective pressure on gut microflora and more effective and should be used in healthcare system as kills selectively susceptible bacterial cells and allowing having least side effects. resistant form to survive. The surviving resistant bacterial grow continuously producing a large population of MATERIALS AND METHODS resistant cells and became predominant in the gut. Such resistant form when release through fecal material in the Samples: One E. coli isolate recovered from morbid environment, spreads the resistance genes to other chicken was processed at the Institute of Microbiology bacterial genera (Shecho et al., 2017). Among the (IOM), University of Veterinary and Animal Sciences pathogens, E. coli is the most lethal pathogen that can be (UVAS), Lahore. Reference bacterial strain of E. coli transmitted to humans directly through sewage (ATCC 25922) was obtained from the IOM, UVAS, contamination of food chain system. Being present as Lahore.

166 167 Pak Vet J, 2021, 41(1): 166-168.

Identification of E. coli: The E. coli isolated from bird DMSO was added and 2-fold serial dilutions were made sample was initially identified based on culturing in up to 10th well. After that, 50µl of 0.5 Mcfarland bacterial tryptone soya broth (TSB) which is an enrichment media, suspension was added in all wells from 1 to 11, while 12th EMB agar plates and biochemical test viz IMViC test well was considered as a negative control. Microtitration (Cappuccino and Sherman, 1999). plate was then incubated at 37ºC for 24 hrs. Bacterial growth was determined by turbidity in negative control Confirmation of E. coli by PCR: Confirmation of E. coli well. The MIC values were determined by observing the was done through PCR. Extracted DNA was subjected to wells with no bacterial turbidity comparing with negative PCR by targeting E. coli specific uspA gene using PCR control well (Shaheen et al., 2015). condition as described by (Shaheen et al., 2015). RESULTS AND DISCUSSION Multi-drug resistance (MDR) testing: The E. coli isolated from the bird was subjected for antibiotic In the present study, ethanolic extracts of seven sensitivity test by Kirby-Bauer method. Fresh bacterial different flowers were evaluated against E. coli (ATCC colonies were suspended in normal saline to produce 25922) and MDR E. coli. The isolate recovered from turbidity equal to 0.5 McFarland units. The suspension morbid chicken showed green metallic sheen on EMB was swabbed on Muller Hinton agar (MHA) using a plates and PCR exhibited corrected band size of 884 bp sterile cotton swab. Antibiotic discs were placed on MHA similar to the study conducted on E. coli isolated from plates and incubated for 24 hrs at 37°C. After incubation, chicken (Sheikh et al., 2012). Results of antibiotic zones of inhibition were measured (mm). sensitivity test revealed that E. coli showed resistance against more than three different classes of antibiotics Antibacterial activity of herbal extracts against bacterial used i.e Ceftazidime, Ofloxacin, Erythromycin, pathogens Doxycycline, Ampicillin, and Augmentin (Fig. 2). In Flowers collection: 100g of dried flowers named as order to observe the antimicrobial potential of flowers, Achillea millefolium, Bombax ceiba, Chrysanthemum initially, various concentrations of ethanolic extracts, cinerarifolium, Hyssopus officinalis, Rosa damascena ranging from 50mg/ml to 400mg/ml, were used as pilot Miller, Taraxacum officinale Weber, Woodfordia study. Out of which concentration of (400mg/ml) was fruticosa were purchased from local herbs market and selected for further testing as at this concentration all transferred to IOM, UVAS, Lahore. extracts showed bacterial inhibition by spot test as well as MIC as shown in Table 1. Preparation of Ethanolic flowers extracts: Ethanolic The MIC of Rosa damascena Miller against E. coli extracts of collected flowers were prepared by grinding (ATCC 25922) and MDR E. coli was 12.5mg/ml and the flowers into fine amorphous powder. Briefly, 10 g of 6.125mg/ml similar to the previous study (Halawani, powder was soaked into 90 ml of 80 percent ethanol (1:10 2014). The antimicrobial effect of Achillea millefolium w/v). After 2 days of incubation in shaking incubator at was achieved at 50mg/ml and 25mg/ml MIC values 150 rpm at 37oC incubation, the extracts were filtered against MDR E. coli and the control, respectively. through filter paper No.1 (Whatman, USA). The extracts Analyzing further, flowers of Bombax ceiba inhibit E. coli were poured into glass petri plates to dry at 40°C. The (ATCC) at 50mg/ml concentration and MDR E. coli at stock solution of dried extracts were prepared by using 10% dimethyl sulfoxide (DMSO) (Obeidat, 2011; 3.125mg/ml comparable to the study on methanolic Shaheen et al., 2015). extracts of B. ceiba stem bark (100µg/disc) which also possess strong antibacterial activity against E. coli Spot test: Antibacterial activity of ethanolic extracts were (Akhtar and Mustafa, 2017). determined by spot test. By internal study, the The ethanolic extract of Woodfordia fruticose Kurtz concentration of each extract was optimized. Initially, flowers inhibit E. coli and MDR E. coli at MIC value of 200mg/ml extract concentration was used and performed 12.5 mg/ml and 6.25 mg/ml, respectively. Similarly, its repeatedly with increased concentration until get clear acetone extract exhibited antimicrobial activity against E. zones. After that, 0.5 McFarland’s bacterial suspensions coli at 300µg/ml concentration. Furthermore, essential oils were used for the preparation of bacterial lawn on MHA of Chrysanthemum coronarium found inhibitory effect plates. A total of 10µl of each extract, having against E. coli ATCC 29425 by disc diffusion method in concentration of 200mg/ml, 250mg/ml, 300mg/ml, contrast to the present study where its flowers had MIC 350mg/ml and 400mg/ml were placed in the center of the value at 50mg/ml and 25mg/ml against E. coli (ATCC) plate and the plate was incubated at 37ºC for 24hrs. After and MDR E. coli, respectively. incubation, the spots showed bacterial inhibition The flower extract of Hyssopus officialnalis showed considered having antibacterial potential and the activity against E. coli ATCC and MDR E. coli at MIC concentration at which all extracts showed antibacterial 50mg/ml and 25mg/ml concentration In a recent study, activity was selected for further processing. nanoparticles of Taxacum officianale leaves also revealed strong antibacterial activity against gram negative Minimum inhibitory concentration (MIC) phytopathogens at various concentrations ranged from Determination: For MIC, broth microdilution method 10ug/ml to 30ug/ml (Saratale et al., 2018) similar to the was used. Briefly, Muller Hinton broth (50 µl) was added present study where Taxacum officinale flowers exhibited in all wells of microtitration plate, from well 1 to 12. Then antibacterial activity with 25 mg/ml MIC against E. coli in first well, 50µl of stock solution of the extract in ATCC and 12.5mg/ml against pathogenic E. coli. 168 Pak Vet J, 2021, 41(1): 166-168.

Table 1: Spot Test and Minimum inhibitory concentration values of herbal extracts SR. No Herbal Extracts Concentration Spot Test MIC E. coli MDR E. coli MDR E. coli (Control) E. coli (Control) 1. Rosa damascena Miller 400mg/ml +ve +ve 1:32 1:128 2. Achilleamillefolium 400mg/ml +ve +ve 1:8 1:16 3. Bombax ceiba 400mg/ml +ve +ve 1:8 1:128 4. Taxacumofficianale weber 400mg/ml +ve +ve 1:16 1:32 5. Woodfordiafruticosakurtz 400mg/ml +ve +ve 1:32 1:64 6. Hyssopusofficianalis L 400mg/ml +ve +ve 1:8 1:16 7. Chrysanthemum cinerafolium 400mg/ml +ve +ve 1:8 1:16

coli except Hyssopus officianalis and Chrysanthemum cinerafolium with each other. In multiple comparisons, independent (I. flower) were compared with dependent (J. flowers). The mean difference showed that the results were also significant (P-value <0.05). The current results reveal that these flowers possess strong antibacterial activity and thus can be used for treatment purpose.

Authors contribution: Conceive and designed the experiments: MR, AAS, SF Performed experiments: IN Analyzed the data: IN, AAS, MIR Wrote paper: MR, IN, AAS, SR.

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